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A riboflavin-producing Bacillus subtilis and its construction method and application

A technology for Bacillus subtilis and riboflavin, which is applied in the field of Bacillus subtilis and its construction, can solve the problems of large workload, difficulty in obtaining riboflavin-producing strains, and limitation of Bacillus subtilis' ability to improve riboflavin production.

Active Publication Date: 2022-07-29
TONGLIAO MEIHUA BIOLOGICAL SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In recent years, although many genetically engineered strains of Bacillus subtilis with high riboflavin production have been obtained through metabolic engineering, due to the limitations of our understanding of microbial physiology and complex metabolic network regulation mechanisms, genetic engineering techniques have been used to continue to transform the strains. It is difficult to obtain strains whose riboflavin production is further greatly improved; and the traditional mutagenesis breeding has a large workload and lacks efficient and rapid screening methods, so that Bacillus subtilis is greatly restricted in improving the riboflavin production capacity. limits

Method used

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  • A riboflavin-producing Bacillus subtilis and its construction method and application
  • A riboflavin-producing Bacillus subtilis and its construction method and application
  • A riboflavin-producing Bacillus subtilis and its construction method and application

Examples

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Embodiment 1

[0029] Example 1. Mutagenesis screening to obtain high-producing strains of riboflavin

[0030] Using Bacillus subtilis 168 (Bacillus subtilis 168) as the original strain, B. subtilis 168 strain was subjected to conventional mutagenesis treatment with ultraviolet 15W, 30cm, 20min, and then nitrosoguanidine was used for mutagenesis under the conditions of 0.4mg / mL, 36℃, 20min. Then, spread on the minimal medium containing 0.2g / L 8-azaguanine (g / L: glucose 20, ammonium sulfate 2, magnesium sulfate 0.4, calcium chloride 0.02, ferrous sulfate 0.02, disodium hydrogen phosphate 1.5. Zinc sulfate 0.01, manganese sulfate 0.01, potassium dihydrogen phosphate 1.5, agar 18, pH 7.0-7.2), cultured at 36°C for 24h. After that, the best growing strain was selected for the next round of mutagenesis and the concentration of 8-azaguanine in the minimal medium was increased. After several rounds of mutagenesis screening, the B.subtilisMHZ-1908-1 strain was obtained, which could grow on 8-azagu...

Embodiment 2

[0032] Example 2: Construction of B.subtilis168, Δupp strain by gene-free editing method

[0033] Taking Bacillus subtilis B.subtilis168 as the starting strain, the gene traceless editing method used in the present invention is based on the two-step integration mediated by thermosensitive plasmids, and is screened through chloramphenicol and 5-fluorouracil (5-FU) reverse screening (Applied Microbiology and Biotechnology, 2014, 98(21):8963-8973. Zhang W, Gao W, Feng J, et al). The screening method needs to firstly delete the upp gene on the genome of the target strain, which encodes uracil phosphoribosyltransferase. When upp and 5-FU coexist, it has a lethal effect on cells.

[0034] The specific construction process is as follows: using primers upp-1f / 1r and upp-2f / 2r, using B.subtilis 168 genome as a template, using pfu DNA polymerase to amplify the upstream and downstream homology arms of 888bp and 938bp, respectively, using primer upp -1f / 2r was amplified to obtain upstrea...

Embodiment 3

[0035] Example 3: Engineering strain B. subtilis 168, Δupp, purR A148D build

[0036] Using the primers purR-1f, purR-1r and purR-2f, purR-2r, using the B. subtilis 168 genome as a template, using pfu high-fidelity DNA polymerase to amplify, respectively, to obtain purR A148D Upper and downstream homology arms; use primers purR-1f and purR-2r to fuse and amplify the upstream and downstream fragments to obtain purR A148D The fusion fragment (containing the A148D mutation, the complete purR wild-type and mutant nucleotide sequences are shown in SEQ ID No. 1 and 2; the specific encoded wild-type and mutant protein sequences are shown in SEQ ID No. 7 and 8. The fusion fragment and the pKSU plasmid (tool vector) were digested with SalI and PstI respectively, then connected and transformed into Trans1 T1 E. coli competent cells. Finally, the recombinant plasmid pKSU-purR was obtained A148D .

[0037] Subsequent transformation and screening methods were the same as those in Exampl...

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Abstract

The invention belongs to the field of microbial fermentation, and discloses a riboflavin-producing Bacillus subtilis and a construction method and application thereof. The Bacillus subtilis of the present invention is a Bacillus subtilis strain with at least one gene locus mutated in purR, ribC or pyrE. The Bacillus subtilis strains obtained by mutating at least one gene site of purR, ribC or pyrE have significantly increased riboflavin accumulation under the fermentation conditions disclosed in the present invention. The Bacillus subtilis of the present invention is a high-producing strain of riboflavin, and the riboflavin produced is significantly increased, laying a foundation for the industrial production of riboflavin.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a riboflavin-producing Bacillus subtilis and a construction method and application thereof. [0002] The riboflavin-producing strains currently used in production include yeast, Bacillus, Corynebacterium and Escherichia coli, and most of them are wild strains or mutagenized strains with poor fermentation performance. Traditional production strains are mainly obtained by multiple rounds of physical and chemical mutagenesis and screening of structural analogs. However, due to the uncertainty of mutation points introduced by random mutagenesis, these strains often have complex genetic backgrounds. The analysis of the role of relevant mutation points will consume a lot of time and energy, which also makes the subsequent metabolic engineering face greater challenges. At the same time, the strains obtained by mutagenesis generally have disadvantages such as difficult t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/54C12N15/75C12P25/00C12R1/125
CPCC07K14/32C12N9/1085C12N9/1077C12N15/75C12P25/00C12Y205/01009C12Y204/0201
Inventor 吴涛胡丹常利斌龚华李岩赵津津
Owner TONGLIAO MEIHUA BIOLOGICAL SCI TECH CO LTD